Various proteins and peptides are useful and valuable for human-health (and other) purposes. For example, there is a broad class of anti-microbial peptides (AMPs) that have, by definition, antibiotic properties. These peptides have the potential for broad use as therapeutics, anti-infectives, disinfectants, and preservatives—if they could be economically produced.
Other types of small peptides include hormones, which also have a wide range of therapeutic uses.
Antimicrobial peptides are natural components of natural antimicrobial defenses of many types of organisms, including mammals, birds, reptiles, insects, and plants. There are many types of antimicrobial peptides, and there are many natural sources of these peptides. Many classes of natural AMPs have been named. For example there are magainins from frogs (See e.g. Zasloff et al. (1987)); attacins and cecropins from silk moths (See e.g. Bowman et al. (1983)); defensins from rabbits, humans, and other mammals (See e.g. Pardi et al. (1992)); indolicidins and bacteriocins from cows (See e.g. Selsted et al. (1992) and Niidome et al. (1999)); and apidaecins from honeybees (See e.g. Casteels et al. (1989)). See also WO 00/31729.
In addition, AMPs are produced by Arthropods (crabs, shrimp, horseshoe crabs, spiders, scorpions) and lower invertebrates (earthworms, mollusks and sponges). Microorganisms also produce AMPs, including myxobacteria, actinomycetes, eubacteria, fungi (both Ascomycetes and Basidiomycetes), and protists such as amoeba. See Table 1.
TABLE 1Author and YearCitationGroup of Organisms(1999)JP 2854872 B2Horseshoe CrabsAszodi et al. (1999)U.S. Pat. No. 5,891,851Actinomycetes(Hoechst AG)Bachere et al. (1999)WO 99/05270 A2ShrimpBowman et (1983)The EMBO Journal 2: 571-576Silk Moths, Attacinsand CecropinsCasteels et al. (1989)The EMBO Journal 8: 2387-2391Honeybees,ApidaecinsCho et al. (1998)Biochimica et Biophysica ActaAnnelids1480: 67-76Gulavita et al. (1996)U.S. Pat. No. 5,516,755Sponges(Harbor Branch OceanographicInstitution)Haeberli et al. (2000)Toxicon 38: 373-380SpidersHetru et al. (2000)Biochemical Journal J 345: 653-664ScorpionsIwanaga et al. (1998)Frontiers in Bioscience 3: D973-84CrabsLeippe et al. (1999)Developmental and ComparativeAmoebaeImmunology 23: 267-79Logeman et al.EP 525508 A2 (Max PlanckBasidiomycetes(1991)Institute)Mitta et al. (1999)Journal of Cell Science 112: 4233-4242MolusksNiidome et al. (1999)Peptide Science 36: 403-406Cows, BactenecinsPardi et al. (1992)Biochemistry 31: 11357-11364Rabbits and Humans,DefensinsRyals et al. (1998)U.S. Pat. No. 5,716,849Myxobacteria(Novartis)Selsted et al. (1992)J. Biological Chemistry 267: 4292-4295Mammals,IndolicidinsUlbrich et al. (1998)U.S. Pat. No. 5,824,874Ascomycetes(Hoechst)Zasloff et al. (1987)PNAS 84: 5449-5453Frogs, Magainins
Some AMPs and structurally similar peptides have been found to have selective activity against cancer cells. See e.g. WO 97/33908 (CSIRO) and WO 90/12866 (Louisiana State University). It has also been possible to synthesize man-made AMP and anti-tumor peptide sequences by modifications of natural analogs (see e.g. U.S. Pat. No. 5,994,306; Intrabiotics), by design from general principles of peptide structure/activity relationships (see e.g. U.S. Pat. No. 5,861,478; Helix Biomedix), or even by screening of random combinations (see e.g. U.S. Pat. No. 5,504,190; Torrey Pines Institute).
Typical AMPs are approximately 10 to 50 amino acids in length. These peptides tend to be relatively rich in basic amino acids (lysine and arginine) and thus tend to be cationic (having a net positive charge). AMPs are amphipathic in nature (i.e., one part of the molecule is hydrophilic while the other part is hydrophobic). Though widely studied, the mode of action of AMPs remains a subject of scientific debate. In many cases the data suggests that the amphipathic peptides organize to form pores or channels in membranes. See e.g. Durell et al. (1992), Biophysical Journal 63:1623-1631. In other cases the AMPs appear to disrupt a membrane by forming a “carpet-like” association with the membrane. See e.g. Shai et al. (1995), Biochemistry 34:11479-88. This disrupts and kills microbes by causing cellular membrane depolarization and the loss of essential cellular components.
AMPs have broad-spectrum antimicrobial activities; this is one attractive aspect of using AMPs as pharmaceutical antibiotics. In light of the increasingly widespread appearance of pathogenic microbes that are resistant to a range of typical chemical antibiotics, there is interest in using antimicrobial peptides (AMPs) as an alternative to typical chemical antibiotics if they could be economically produced.
AMPs have the potential for broad use as therapeutics (see e.g. U.S. Pat. No. 6,132,775; New York University), anti-infectives (see e.g. U.S. Pat. No. 6,071,879; University of Oklahoma), disinfectants (see e.g. Jaynes et al. [1996], CLAO J. 22:114-7), preservatives (see e.g. WO 00/01400; Assoc. Cape Cod Inc), and for food safety (see e.g. Padgett et al. [1998], Journal of Food Protection 61:1330-1335). AMPs and AMP-like peptides are also of interest for therapeutic uses against cancer and viruses (see e.g. Egal et al. [1999], International Journal of Antimicrobial Agents 13:57-60), including retro-viruses (see e.g. Tamamura et al. [1998], Bioorganic and Medicinal Chemistry 6:231-238). There is also considerable interest in using AMPs for the control of plant diseases, primarily through using transgenic plant approaches. See, e.g., Norelli et al. (1999), Phytopathology 89:S56.
However, a practical limitation to large-scale therapeutic and related uses of AMPs or other short peptide sequences is that they are expensive (and difficult/inefficient) to produce in mass quantities. For example, chemical peptide synthesis of AMPs (and other peptides or proteins) is very costly.
The synthetic production of heterologous proteins of therapeutic or functional (e.g., catalytic) significance in microorganisms has been attained. See e.g. Swartz, J. R. (2001), Current Opinion in Biotechnology 12:195-201. Such methods of producing polypeptides have the potential for providing some advantages over solid phase synthesis, including sequence fidelity, convenience, low cost, and the ability to produce long polypeptides/proteins. While microbial production of certain types of proteins and polypeptides can be convenient and cost-effective, such techniques cannot be universally applied, and limitations are often evident. These limitations can include: 1) low yield, 2) accumulation of misfolded and inactive protein, 3) inhibition of microbial growth, and 4) difficulties with detection or isolation/purification of polypeptides, particularly those of low molecular weight.
Attempts have been made to biologically produce small peptides as part of larger fusion proteins to improve overall protein yield. For example, U.S. Pat. Nos. 6,242,219 and 6,274,344 (Xoma Corp.) relate to a peptide derived from a bactericidal/permeability-increasing protein (BPI) fused to (and cleavable from) a carrier protein. The '219 patent relates to simultaneous acid lysis and cleavage of the peptide from the carrier. Asp-Pro linkage can be used between the peptide and the carrier. The '344 patent indicates that it might be possible for the carrier to be cationic, like the BPI.
Small peptides are quite susceptible to degradation by native proteases in bacteria. Small peptides, including AMPs, may be produced in nature as part of a multipeptide precursor. Casteels-Josson et al. (1993), EMBO Journal, 12(4):1569-1578. Insect neuropeptides (short peptides) appear to be produced naturally in a like manner. See, e.g., Rao et al. (1996), Gene 17:1-5. However, it can be quite difficult to replicate natural events in vitro. For example, assembling and expressing multiple copies of a desired DNA fragment in an easily cleavable manner can be a laborious and costly process. For background on amplifying multiple copies of cloned DNA segments, more generally, see e.g. Cohen et al. (1986), DNA 5(4):334-345, which relates to the use of tandem repeats of DNA fragments to produce multimers having inverted repeat structures (polyoma virus DNA was used as the monomer); and Kim et al. (1988), Gene, 71:1-8, which relates to the amplification of cloned DNA as tandem multimers (wherein the monomers have asymmetric cohesive ends).
Thus, there are hurdles to the synthetic production of small peptides, and AMPs are particularly difficult to synthesize biologically. In addition to being short and subject to protealytic degradation, AMPs are, by definition, toxic to bacteria and other microbes. Thus, production of them in bioreactors as native, active material is practically precluded. Furthermore, AMPs tend to be positively charged/cationic, which presents other obstacles to synthetic biological production as explained in more detail below.
Recombinant E. coli, for example, have been used in attempts to biosynthetically produce AMPs. However, the yields (heretofore), if any, have been extremely low. By definition, AMPs are toxic to bacteria and other microorganisms. Thus, when any significant amount of AMP is produced by a culture (of E. coli, for example), the AMPs tend to kill the cultures. U.S. Pat. No. 5,206,154 (Xoma Corporation) claims a cecropin fused to a carrier (araB) in an effort to suppress the activity of the cecropin. If this approach was successful, the relative yield of the AMP, relative to the carrier, would be low.
Attempts have also been made to synthetically produce AMPs by first producing them as a multimeric/concatemeric precursor that is later cleaved to yield the active monomers. However, there were also problems with producing AMPs in that manner.
Attempting to produce AMPs as a multipeptide precursor poses unique, especially difficult problems with biological synthesis. More specifically, because of the highly cationic/positive charge of a single AMP, creating a multimeric protein comprising a plurality of AMP monomers is essentially creating a larger, highly cationic protein. It has been proposed that the positive charge of the nascent AMP multimer (which is cationic due to the abundance of basic amino acid residues in the AMPs), interacts with the negative charge of the DNA and/or RNA (which are acidic) involved in transcription and translation, thereby disrupting natural cellular processes and preventing production of the desired AMP to any substantial level. See, e.g., Lee et al. (1996), Genetic Analysis: Biomolecular Engineering, 13:139-145 (relating to the amplification of constructs comprising tandem multimers of 93 basepair magainin monomers; it is proposed therein that positively charged amino acids of the polymer should be neutralized by fusing the polymer to a negatively charged protein). Protease degradation of product peptides is another observed problem.
Various attempts have been made to produce AMPs in the form of fusion proteins in which the positive charge of the AMP is balanced or neutralized by a carrier protein. WO 96/28559 (University of British Columbia; Hancock et al.) relates to a fusion protein having an anionic AMP portion and a cationic/LPS-binding portion that is said to suppress the anti-microbial activity of the cationic portion.
U.S. Pat. No. 5,593,866 (University of British Columbia) describes the use of a cationic/anionic fusion in an attempt to biosynthetically produce AMPs. This patent discloses a cecropin/melittin fusion comprising the first 18 amino acids of cecropin and the last 8 amino acids of melittin. Examples of the carrier peptides described therein include the GST protein from S. aureus and an outer membrane protein, protein F, from P. aeruginosa. Cyanogen bromide was used to cleave the fusion to recover active peptides. This patent claims the use of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa for producing the fusion protein. This patent also states that the AMP of that invention can be used to inhibit E. coli, P. aeruginosa, E. cloacae, S. typhimurium, and S. aureus. This patent also relates to an AMP having two additional lysine residues on the carboxy terminus; this was reported to have surprisingly doubled the antimicrobial activity of the AMP.
Lee et al. (1998), J. Microbiol. Biotechnol., 8(1):34-41, use a maltose-binding protein fused to magainin multimers in an attempt to achieve adequate expression. They used Factor Xa cleavage sites between the monomers. This reference stated that the AMPs resulting from the Factor Xa cleavage surprisingly retained activity despite having additional amino acid residues on the core AMP.
Lee et al. (February 1998; Protein Expression and Purification, vol. 12, no. 1, pp. 53-60) use tandem repeats of an acidic peptide fused to the basic/positively charged AMP (buforin II). A cysteine residue was added as a critical element to each end of the acidic peptide. Cyanogen bromide was the cleavage agent used therein.
Lee et al. (June 1999), J. Microbiol. and Biotech., 9(3):303-310, use a buforin II AMP fused to a cysteine-rich acidic peptide. Zhang et al. (1998), Biochemical and Biophysical Research Communications, 247:674-680, relates to fusion proteins comprising a cecropin/magainin and a carrier protein comprising an anionic prepro domain, a RepA domain, and a cellulose-binding domain.
WO 98/54336 (Kim, Lee, et al.; Samyang Genex and Korea Advanced Institute Science; see also U.S. Pat. No. 6,183,992) relates to the use of a fusion of an AMP (Buforin II) with an acidic peptide (Guamerin) that has at least two cysteine residues. This application teaches that the acidic peptide is required to neutralize the basic/positively charged AMP in order to prevent electrical attractions and interactions with DNA and RNA during translation of the AMP. The cysteine residues are also taught to be necessary to facilitate the interaction and proper folding of the two portions of the polypeptide. WO 98/54336 distinguishes U.S. Pat. No. 5,593,866 by stating that a general acidic carrier peptide gene alone does not permit an efficient expression of a basic antimicrobial peptide; the presence of at least two cysteine residues in the acidic peptide is also needed to efficiently solve the problem. WO 98/54336 similarly describes WO 96/28559 as being inoperative and likewise suggests the use of cysteine residues in the anionic peptide as the solution to the problem.
WO 99/64611 (Samyang Genex Corp.) relates to the use of fusions comprising a purF gene and an AMP. Another Samyang Genex application, WO 00/34312, relates to the use of hydroxylamine for cleaving a basic peptide/protein from a fusion partner. U.S. Pat. No. 6,255,279 and WO 99/48912 (Korea Advanced Institute) mention the possible use of an AMP in mouthwashes and eyewashes. WO 97/22624 (Beiersdorf AG) relates to the use of random multimers for use in an antimicrobial cosmetic preparations, deodorants, and the like.
WO 00/31279 (Micrologix Biotech Inc.) relates to a “multi-domain fusion protein,” which appears to be a multimeric AMP fused to an acidic peptide (a cellulose-binding domain) wherein there are, as an essential element, anionic spacers between each AMP monomer for “charge balancing” (i.e., to eliminate the charge of the cationic peptide component). This application uses small spacers (that provide a concentrated negative charge) to raise the relative production of the AMPs as compared to total protein produced by the cell; this type of multimer can itself be fused to a carrier protein (a cellulose-binding domain). Although this application briefly mentions that 70% formic acid might be a possible cleavage agent, cyanogen bromide (CNBr) is exemplified throughout this application to cleave (at methionine residues) the multi-domain protein to yield active monomers. WO 00/31279 states that the fusion protein can be an insoluble protein. An “insoluble peptide” is defined therein as “a polypeptide that, when cells are broken open and cellular debris precipitated by centrifugation (e.g., 10,000×g to 15,000×g), produces substantially no soluble component, as determined by SDS polyacrylamide gel with Coomassie Blue staining.”
There has been no suggestion in the art that Pseudomonas fluorescens (P. fluorescens) would be advantageous for or capable of producing AMPs.
Pseudomonas aeruginosa and P. fluorescens have been used commercially for the production of Vitamin B12. See Schenectady County Community College website. Certain strains of P. fluorescens are known to have anti-fungal properties. See, e.g., U.S. Pat. No. 6,048,713. Some strains of P. fluorescens are known to produce antibiotics and can be used in the production of these antibiotics. See, e.g., U.S. Pat. No. 4,108,724. See also U.S. Pat. No. 5,348,742. The use of P. fluorescens to produce insecticidal protein toxins is also known. See, e.g., U.S. Pat. Nos. 5,840,554; 5,527,883; 5,128,130; and 5,055,294 (Mycogen Corporation). P. fluorescens has also been used for bioremediation of environmental contamination. See, e.g., U.S. Pat. Nos. 5,711,945 and 4,853,334.
In all of these peptide expression systems, the yield of polypeptide expressed from the transgene in the cell culture is typically reported in the range from a few micrograms per liter to about 100 mg/L. As a result, there is still a need in the art for transgenic polypeptide expression systems that provide significantly higher yields. Thus, there is a long-felt and critical need for methods of producing small peptides, including AMPs, in efficient, cost-effective manners using microbial fermentation.